Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Authors:
Dityateva G, Hammond M, Thiel C, Ruonala MO, Delling M, Siebenkotten G, Nix M and Dityatev A
In:
J Neurosci Methods (2003) 130(1): 65-73
Research Area:
Neurobiology
Cells used in publication:
Neuron, hippocampal, chicken
Species: chicken
Tissue Origin: brain
Neuron, hippocampal, mouse
Species: mouse
Tissue Origin: brain
Experiment
Dissociated neonatal mouse and embryonic chicken hippocampal neural cells were tested: - Nucleofection conditions with eGFP were optimized and examined by immunofluorescence microscopy in time-course experiments. -Co-localization of eGFP and DsRed were revealed by co-transfection. -Neurite outgrowth and interaction, cell development and localization of synaptic markers were monitored for 7 days and visualized by time-lapse video recording. -Cell surface expression of NCAM140 was examined in neurons from NCAM deficient mice. -Neuronal lipid rafts were labeled by GPI-eGFP expression and cells were analyzed for their electrophysical properties.
Abstract
Non-viral gene transfer into neurons has proved to be a formidable task. Here, we describe an electroporation-based method that allows efficient and reliable DNA transfer into dissociated neural cells before they are plated and cultured. In hippocampal neural cells derived from either neonatal mouse or embryonic chicken brains, a high transfection rate was already observed 5 h after transfection, and reached 40-80% in 24 h, as monitored by expression of enhanced green fluorescent protein (eGFP). The level of eGFP expression per cell depended on the amount of DNA used in a gene transfer experiment. The survival and neuritic length of transfected cells resembled that of non-electroporated cells. The transfected neurons showed normal immunostaining for endogenous synaptic protein synaptophysin and the neural cell adhesion molecule (NCAM). Furthermore, efficient gene transfer of the NCAM isoform NCAM140 and eGFP-tagged NCAM140 could be achieved, allowing visualization of NCAM140 expression. Also, a glycosylphosphatidylinositol-anchored eGFP could be efficiently expressed, highlighting lipid rafts without altering electrophysiological properties of transfected neurons. When neurons transfected with green and red fluorescent proteins were cocultured, fine details of their interactions could be revealed in time-lapse experiments. Thus, the method provides a useful tool for elucidation of genes involved in different neuronal functions, including neurite outgrowth, synaptogenesis and synaptic transmission.